ABSTRACTAutophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content; however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

Figure 2: Myosin VI is required for autophagosomal degradation of huntingtin and p62-positive protein aggregates(a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 hours. Cells were processed for immunofluorescence microscopy following 16 hours MG132 treatment (zero time point) and 8 hours post-washout of inhibitor. Immunolabelling for p62 was performed to visualize aggregates (red) and Hoechst was utilized to identify nuclei (blue). Scale bar, 20 μm (b) Quantitation of immunofluorescent p62 positive aggregates was evaluated using an automated Cellomics VTi microscope. Results were calculated as the average p62 fluorescence intensity at 8 hours post-MG132 washout normalized to the zero time point and represented as percent of control (+/− s.d.) (n=3). * p<0.05, ***p<0.001. (c) Parental or stable expressing siRNA resistant GFP-myosin VI Hela cells were transiently transfected with a single target myosin VI siRNA oligonucleotide and were subsequently treated with 1 μM MG132 for 16 hours. Cells were processed for immunofluorescence microscopy at 16 hours post-MG132 treatment (t=0) or allowed to recover following washout of MG132 for 8 hours (t=8). Cells were processed for quantitation with the automated Cellomics VTi microscope to evaluate p62 fluorescence intensity. Results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared to mock control cells following recovery from MG132 washout (t=8) (+/− s.d) (n=3). (d) Hela cells with stable expression of HttQ72-GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy. Immunolabelling was performed for GFP (green) and p62 (red). Nuclei are labelled with Hoechst (blue). Scale bar, 20 μm. (e) Quantitation of HttQ72-GFP aggregates was performed on myosin VI siRNA transfected Hela cells. Results were calculated as the percentage of GFP expressing cells with greater than 15 GFP-positive spots/cell. Results represent the mean (+/− s.d) from n=3 independent experiments, *** p<0.001.

Mentions:
Next, we investigated whether myosin VI dysfunction has an impact on autophagy-dependent clearance of ubiquitinated cargo. RPE cells were treated with MG132 for 16 hours to allow for bulk accumulation of endogenous ubiquitinated protein aggregates19 and 8 hours after washing out this inhibitor, the remaining protein aggregates were visualised with antibodies to p62 (Figure 2a) and quantified by automated microscopy (Figure 2b). Loss of myosin VI using either a pool of four different siRNAs or a single siRNA causes a significant increase in p62-positive aggregates similar to that observed with the knockdown of Atg5, one of the essential autophagy regulators (Figure 2b). This defect in aggregate clearance was rescued by expression of a siRNA resistant myosin VI construct (Figure 2c).

Figure 2: Myosin VI is required for autophagosomal degradation of huntingtin and p62-positive protein aggregates(a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 hours. Cells were processed for immunofluorescence microscopy following 16 hours MG132 treatment (zero time point) and 8 hours post-washout of inhibitor. Immunolabelling for p62 was performed to visualize aggregates (red) and Hoechst was utilized to identify nuclei (blue). Scale bar, 20 μm (b) Quantitation of immunofluorescent p62 positive aggregates was evaluated using an automated Cellomics VTi microscope. Results were calculated as the average p62 fluorescence intensity at 8 hours post-MG132 washout normalized to the zero time point and represented as percent of control (+/− s.d.) (n=3). * p<0.05, ***p<0.001. (c) Parental or stable expressing siRNA resistant GFP-myosin VI Hela cells were transiently transfected with a single target myosin VI siRNA oligonucleotide and were subsequently treated with 1 μM MG132 for 16 hours. Cells were processed for immunofluorescence microscopy at 16 hours post-MG132 treatment (t=0) or allowed to recover following washout of MG132 for 8 hours (t=8). Cells were processed for quantitation with the automated Cellomics VTi microscope to evaluate p62 fluorescence intensity. Results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared to mock control cells following recovery from MG132 washout (t=8) (+/− s.d) (n=3). (d) Hela cells with stable expression of HttQ72-GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy. Immunolabelling was performed for GFP (green) and p62 (red). Nuclei are labelled with Hoechst (blue). Scale bar, 20 μm. (e) Quantitation of HttQ72-GFP aggregates was performed on myosin VI siRNA transfected Hela cells. Results were calculated as the percentage of GFP expressing cells with greater than 15 GFP-positive spots/cell. Results represent the mean (+/− s.d) from n=3 independent experiments, *** p<0.001.

Mentions:
Next, we investigated whether myosin VI dysfunction has an impact on autophagy-dependent clearance of ubiquitinated cargo. RPE cells were treated with MG132 for 16 hours to allow for bulk accumulation of endogenous ubiquitinated protein aggregates19 and 8 hours after washing out this inhibitor, the remaining protein aggregates were visualised with antibodies to p62 (Figure 2a) and quantified by automated microscopy (Figure 2b). Loss of myosin VI using either a pool of four different siRNAs or a single siRNA causes a significant increase in p62-positive aggregates similar to that observed with the knockdown of Atg5, one of the essential autophagy regulators (Figure 2b). This defect in aggregate clearance was rescued by expression of a siRNA resistant myosin VI construct (Figure 2c).

Bottom Line:
Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy.We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion.We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

ABSTRACTAutophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content; however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.